CLONING AND SEQUENCE-ANALYSIS OF THE HEMB GENE OF STAPHYLOCOCCUS-AUREUS

The hemB gene is a member of the family of genes encoding enzymes of t he porphyrin biosynthetic pathway and codes for the enzyme porphobilin ogen synthase, which is responsible for the conversion of Delta-aminol evulinic acid to porphobilinogen. To clone the hemB gene of Staphyloco ccus aureus we used Tn917-mediated transposon mutagenesis. Tn917 confe rs resistance to erythromycin and is carried by plasmid pTV1ts, which has thermosensitive replication. Hem mutants were selected by growth i n the presence of kanamycin and erythromycin at 43 degrees C. Prelimin ary identification of the hem mutants was based on their dwarf colony growth, which could be restored to normal by hemin. DNA extracted from one of the hem mutants was digested with several restriction endonucl eases and hybridized to a probe representing the XbaI-AvaI end of Tn91 7. A Bg/II-EcoRI fragment of 4.5 kb gave a positive signal and was clo ned into pUC18. Transformants were identified by colony hybridization with the Tn917 probe. The positive clones were sequenced, starting fro m the transposon end. The results allowed us to identify an open readi ng frame whose nucleotide sequence presented a homology of 63% to the sequence of the hemB gene of Bacillus subtilis and of 55% to the seque nce of the hemB gene of Escherichia coli K12. No other nucleotide sequ ences, except those belonging to known hemB genes, presented significa nt homologies to our sequence. The cloning of the hemB gene of S. aure us was confirmed by the ability of the gene to complement a hemB mutan t of E. coli K12. To our knowledge, this is the first report of the cl oning of a hem gene in S. aureus.